KR20010023870A - Vanadium complexes of monohydroxamates and pharmaceutical compositions comprising them - Google Patents

Abstract

The present invention relates to a monohydroxyxamate vanadium complex of formula (I) useful for inducing normal blood glucose and / or lowering blood glucose concentrations in diabetic patients:

R-CO-NHOH.X

Where

R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n -where n is 1, 2 or 3 and Y is OH or NH ₂ , (ii) H ₂ N-CH ( COOH) -CH ₂ -S-CH ₂ -and (iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl; X is a vanadium compound selected from vanadil (VO ²⁺ ), metavanadate (VO ₃ ⁻ ) or vanadate (VO ₄ ^3- ) salts.

Description

Vanadium complexes of monohydroxyxamate and pharmaceutical compositions comprising the same {VANADIUM COMPLEXES OF MONOHYDROXAMATES AND PHARMACEUTICAL COMPOSITIONS COMPRISING THEM}

Intensive studies of the insulin effect of vanadium on insulin have been undertaken over the past 20 years (Shechter et al., 1995). In vitro, vanadium salts mimic the effects of most insulin on the hormone's major target tissues, and in vivo they induce normal blood glucose and improve glucose homeostasis in insulin deficient and insulin resistant diabetic rodents. Brichard and Henquin, 1995]. In the field of basic research, data suggesting that vanadium exhibits their insulin-like metabolic effects through alternative pathways that do not include insulin-receptor tyrosine kinase activation and phosphorylation of insulin-receptor substrate 1 (IRS-1). Are accumulating. An important function of this alternative system is believed to include inhibition of protein-phosphotyrosine phosphatase and activation of non-receptor protein tyrosine-kinase (Brichard and Henquin, 1995).

Vanadium is an ultratrace element present in the mammalian body. Dietary intake is 10-60 μg per day and intracellular concentration is about 20 nM (Shechter et al., 1995 and Brichard and Henquin, 1995). Most intracellular vanadium will be in vanadil (+4) form. Acute oral administration of vanadium compounds has proven quite detrimental. Nevertheless, the significant antidiabetic effects of oral vanadium treatment in insulin deficient and insulin resistant diabetic rodents (Brichard and Henquin, 1995) have encouraged the start of clinical trials. Small doses of vanadium (100-125 mg / l / day, period over 3 weeks) were allowed, which was 100 times lower than the dose used in most animal experiments, but some beneficial effects were observed. Cohen et al., 1995.

Organic chelated vanadium (+4) complexes are advantageous in promoting the metabolic effects of insulin in vitro [Li et al., 1996] and in vivo [Sakurai et al., 1995] in STZ-rats. About five times more potent than vanadium (+4). The theoretical basis for this is still under study, but is believed to be the result of stabilizing more potent insulin-like vanadium species.

Vanadium salts mimic the metabolic effects of insulin through alternative insulin-independent mechanisms in vitro and can overcome the state of insulin resistance in diabetic rodents in vivo. Vanadium therapy is a useful, interesting, and complementary method for insulin therapy if vanadium salts are less harmful, or specific treatments can be made to perform vanadium therapy with little or no exogenous vanadium source. do.

Israel Patent No. 99666 and corresponding Applicant's U.S. Patent No. 5,338,759 have the formula R ² R ³ C {CH ₂ O (CH ₂ ) _m CO [NHCHR (CH ₂ ) _q CO], which is mentioned as useful in the treatment of diabetes. _Although vanadil complexes of dihydroxamate of _n NOHR ¹ } ₂ are described, these complexes are subsequently found by the inventors to be inadequate for use in vivo to normalize blood glucose concentrations in model diabetic rats. lost.

Hydroxamic acid derivatives have been found to be involved in the microbial transport of iron, and thus have been proposed for the treatment of iron deficiency diseases. They are also inhibitors of urease and have been proposed for the treatment of hepatic coma. Most of their biological activity is associated with their efficacy in chelating various metals. In the case of metal chelates formed by the majority of hydroxamic acids, coordination occurs after carbonyl oxygen and deprotonated OH coordinate (O, O) after the OH group is deprotonated.

Amino acid monohydroxyxamate is a simple nontoxic derivative of amino acids. D-aspartic acid β-hydroxyxamate (D-Asp (β) HXM) has been shown to have antitumor activity against murine leukemia L5178Y, both in vitro and in vivo, and in vitro It is active against leukemia cells (Tournaire et al., 1994). L-glutamic acid (γ) -monohydroxamate (Glu (γ) HXM) is cytotoxic to leukemia L1210 cells in vitro and cytotoxic to leukemia L1210 and melanoma B16 cells in vivo [Vila et al. al., 1990].

The present invention relates to a vanadium complex of monohydroxyxamate and a pharmaceutical composition comprising the same useful for treating diabetes.

Abbreviations: Asp (β) HXM, L-aspartic acid β-monohydroxyxamate; CytPTK, cytoplasmic protein-tyrosine kinase; Glu (γ) HXM, L-glutamic acid γ-monohydroxamate; HXM, monohydroxyxamate; InsRTK, insulin receptor tyrosine kinase; IRS-1, insulin receptor substrate 1; PTK, protein tyrosine kinase; KRB, Crabs Ringer Bicarbonate; NaVO ₃ , sodium metavanadate; STZ, streptozosin; VOSO ₄ , vanadil sulfate; VOCl ₂ , vanadil chloride.

1 compares the concentration dependent activation of lipoogenesis by 1: 1 to 1: 5 complexes of Glu (γ) HXM: VOCl ₂ (+4) with free VOCl ₂ and free Glu (γ) HXM.

FIG. 2 shows that the 1: 1 complex of Glu (γ) HXM: VOCl ₂ (+4) increases the normal blood glucose effect of vanadium in STZ rats compared to free VOCl ₂ .

FIG. 3 shows concentration dependent activation of lipid production by Glu (γ) HXM: NaVO ₃ (1: 1 complex), free NaVO ₃ and free Glu (γ) HXM.

FIG. 4 shows 2: 1 complexes of free Glu (γ) HXM, free NaVO ₃ and Glu (γ) HXM: NaVO ₃ stimulate hexose influx at two different concentrations.

FIG. 5 shows that Glu (γ) HXM: NaVO ₃ (2: 1 complex) lowers blood glucose concentration compared to free Glu (γ) HXM and free NaVO ₃ .

FIG. 6 shows that free Glu (γ) HXM activates lipid production in rat adipocytes in the absence of externally added vanadium.

FIG. 7 shows the degree of lipid production induced by free Glu (γ) HXM, free NaVO ₃ or insulin with increasing concentration of staurosporin.

FIG. 8 is a comparison of lipoogenesis activation induced by increasing the concentration of free Glu (γ) HXM in normal adipocytes and vanadium rich adipocytes.

9 shows the lipidogenic ability of dry powders of free Glu (γ) HXM, VOCl ₂ and NaVO ₃ , and Glu (γ) HXM: VOCl ₂ and Glu (γ) HXM: NaVO ₃ 2: 1 complexes stored at room temperature. It is a figure compared.

Description of Preferred Embodiments

According to the present invention, the only specific increase in fetal efficacy of vanadium insulin is achieved by the specific monohydroxyxamate complex of vanadium. In particular, L-glutamic acid (γ) monohydroxyxamate (Glu (γ) HXM) increases the efficacy of vanadium in activating glucose metabolism in rat adipocytes by about 7 to 10 times, and in vivo STZ treated diabetes mellitus. The effectiveness of vanadate in lowering blood glucose concentrations in rats is increased five to seven times. The increase in efficacy is greatest at a 2: 1 molar ratio of L-Glu (γ) HXM: vanadium. Both the unmodified α-amino and α-carboxyl portions of L-Glu (γ) HXM are essential for increasing efficacy. Moreover, the synergy of L-Glu (γ) HXM is stereospecific and not promoted by D-Glu (γ) HXM, but D-Glu (γ) HXM also complexes with vanadium. Interestingly enough, of all the described vanadium complexes that increase the insulin action of vanadium, L-Glu (γ) HXM is also unique in that it activates lipoogenesis in rat adipocytes in the absence of externally added vanadium. This effect appears through the vanadium pathway by experimental data suggesting that L-Glu (γ) HXM can convert physiologically trace amounts of vanadium endogenously present in rat adipocytes into fetal active species of insulin. It is further demonstrated herein. Physicochemical experiments of these active complexes exhibit unique physicochemical characteristics. Vanadium is also present in the +5 oxidation state at physiological pH values, while in equilibrium with this when prepared with vanadil +4 cations.

In vitro screening assays used in the present invention show that in addition to Glu (γ) HXM, L-Asp (β) HXM and nicotinic acid-HXM (1: 1 molar ratio) also increase the insulin potency of vanadium (+4). Indicates. Their synergistic effect is about 85% and about 57% of the effect obtained by Glu (γ) HXM. In contrast, the D-isomers of both Glu (γ) HXM and Asp (β) HXM as well as α-amino acid hydroxamate did not increase the fetal efficacy of VOCl ₂ insulin.

The invention will now be illustrated by the following non-limiting examples.

Experiment process

(a) Material. D- [U- ¹⁴ C] glucose and 2-deoxy-D- [G- ³ H] glucose were purchased from New England Nuclear, Boston, Mass. Collagenase type I (134 U / mg) was obtained from Worthington Biochemicals, Freehold, NJ. Porcine insulin was purchased from Eli Lilly Co., Indianapolis, IN. Fluoretine, 2 deoxyglucose, L-glutamic acid γ-monohydroxamate (Glu (γ) HXM), glycine hydroxyxamate (Gly-HXM), L-isoleucine hydroxamate (isoleu-HXM), L- Tryptophan hydroxamate (Trp-HXM), L-tyrosine hydroxyxamate (Tyr-HXM) and L-cystine dihydroxamate (cystine HXM ₂ ) were obtained from Sigma Chemical Co., St. Louis, Mo. Purchased from.

Craps-Ringer bicarbonate (KRB) buffer (pH 7.4) contained 110 mM NaCl, 25 mM NaHCO ₃ , 5 mM KCl, 1.2 mM KH ₂ PO ₄ , 1.3 mM CaCl ₂ , 1.3 mM MgSO ₄ .

All other chemicals and reagents used in this experiment were of analytical grade.

(b) Streptozosin (STZ) treated rats: Diabetes was induced by a single intravenous injection (155 mg / kg by weight) of a freshly prepared solution of streptozosin in 0.1 M citrate buffer (pH 4.5). The effect of the tested compound on blood glucose concentration was measured 14 days after induction of diabetes.

(c) Cell Preparation and Lipogenesis Bioassay: Rat adipocytes were prepared essentially by the method of Rodbell (1964). The fat meatballs of male Wistar rats were cut into small pieces using scissors and suspended in 3 ml of KRB buffer. The degradation was digested with collagenase (type 1, 134 units / mg; 1 mg / ml) in a 25 ml flexible plastic bottle under an atmosphere of carbogen (95% O ₂ , 5% CO ₂ ). The mixture was vigorously shaken for 40 minutes at < RTI ID = 0.0 > Cell preparations showed over 95% viability by trypan blue exclusion at least 3 hours after degradation. Then 5 ml of buffer was added and the cells passed through a mesh screen. The cells were then suspended while standing in a 15 ml plastic test tube for several minutes at room temperature and the lower buffer was removed. This process (suspension, suspension, removal of lower buffer) was repeated three times.

In the lipidogenesis assay, for determination of glucose uptake and its incorporation into lipids (lipidogenesis), the adipocyte suspension (3 × 10 ⁵ cells / ml) is divided into plastic vials (0.5 ml per vial) and carbogen Incubated at 37 ° C. for 60 minutes in the presence of insulin (100 ng / ml), and 0.2 mM [U- ¹⁴ C] glucose (4-7 mCi / mol) and the complex to be tested. Lipid formation was terminated by addition of toluene base scintillation fluid (1.0 mL per vial) and radioactivity of the extracted lipids was calculated (Moody et al., 1974). In a typical experiment, insulin stimulated lipid production was four to five times higher than baseline. The V base is about 2000 cpm / 3 × 10 ⁵ cells / h; V insulin is about 8000-10,000 cpm / 3 × 10 ⁵ cells / h.

Summary of the Invention

Certain amino acid monohydroxyxamate (HXM), especially L-type glutamic acid γ-monohydroxamate (Glu (γ) HXM) and aspartic acid β-monohydroxyxamate (Asp (β) HXM) are vanadium (+4) And vanadium (+5) have now been found by the present invention. In 1: 1 or 2: 1 HXM: vanadium molar stoichiometry, they mainly increase the fetal efficacy of vanadium (+4) and (+5) insulin ex vivo and in vivo in streptozocin treated rats Normalize glucose concentration.

The present invention relates to vanadium complexes of novel monohydroxyxamates of formula (I):

R-CO-NHOH.X (I)

Where

R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n- ,

(ii) H ₂ N—CH (COOH) —CH ₂ —S—CH ₂ — and

(iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl, wherein n is 1, 2 or 3 and Y is OH or NH ₂ ;

X is a vanadium compound selected from vanadil (VO ²⁺ ), metavanadate (VO ₃ ⁻ ) or vanadate (VO ₄ ^3- ) salts.

According to the invention, for the monohydroxyxamate of (i), n is preferably 1 or 2, i.e., β- and γ-monohydroxamate of L-aspartic acid and L-glutamic acid, respectively. In the most preferred embodiment, the amino acid monohydroxyxamate is Glu (γ) HXM, which has been found to be more effective in demonstrating the metabolic effects of insulin in rat adipocytes compared to many α-amino acid monohydroxyxamates. .

Interestingly enough, among all known vanadium chelators described in the literature, such as acetylacetonate, bispicolinato and dihydroxamate RL-252 described in US Pat. No. 5,338,759, the amino acid monohydroxyxamate is external It is unique in that it has the function of producing an insulin effect ex vivo without added vanadium, which converts a small amount of intracellular amino acid monohydroxyxamate (+4, about 20 nM) into an endogenous species of insulin. It can be made. Further experiments in vitro revealed that Glu (γ) HXM promotes all physiologically related bioeffects of insulin. These include activation of hexose uptake and inhibition of isoproterenol mediated lipolysis. Very importantly, activation by Glu (γ) HXM enhances the maximum effect produced by the saturated concentration of insulin.

Among the monohydroxyxamates of the above (iii), preference is given to 3-pyridyl radicals, ie nicotinic acid hydroxamate, 2- or 3-piperidyl radicals and 3-tetrahydroisoquinolinyl radicals.

The monohydroxyxamates used in the present invention are water soluble in contrast to the dihydroxyxamate of the aforementioned US Pat. No. 5,338,759 which is water insoluble. Thus, the vanadium complex of the present invention can be prepared by simply dissolving the monohydroxyxamate and the vanadium salt in water.

Examples of vanadium salts used to form the complexes used in the compositions of the present invention include VOCl ₂ (+4), VOSO ₄ (+4), NaVO ₃ (+5) and Na ₃ VO ₄ (+5). It is not limited to.

While various HXM: vanadium salt stoichiometric molar ratios of the complexes are contemplated by the present invention, molar ratios of 1: 1 and 2 HXM: 1 vanadium salt are preferred.

The complex of formula (I) of the present invention is prepared by mixing an aqueous solution of monohydroxyxamate and vanadium salt and freeze-drying the solution, for example to obtain a dry powder that can be stored at room temperature.

The present invention also provides a pharmaceutical composition useful for treating diabetes, in particular, for lowering blood glucose levels and inducing normal blood glucose in diabetic patients, the vanadium complex of monohydroxyxamate of formula (I) as an active ingredient and, optionally, Provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. Such compositions can be used for the treatment of both insulin dependent diabetes mellitus (IDDM) and insulin independent diabetes mellitus (NIDDM).

Dosage depends on the condition of the diabetic and can be between 0.2 mg / kg and 2 mg / kg per day. Since the allowed amount of vanadium to be administered to diabetic patients in the current clinical trials is about 2 mg / kg / day, the minimum amount provided by the present invention represents a 10-fold increase in efficacy.

Compositions of the present invention comprising a vanadium complex of formula (I) may be provided in soluble form, for example in the form of a drop or capsule or tablet, preferably administered orally. These compositions may be administered alone or in combination with insulin.

Vanadium complexes of formula (I) can also be produced in vivo by separately administering vanadium salts and monohydroxyxamate. Accordingly, the present invention relates to the formula R-CO-NHOH, wherein R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n- , (ii) H ₂ N-CH (COOH) -CH _2- S-CH ₂ -and (iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl, wherein n is 1, 2 or 3 and Y is OH or NH ₂ A pharmaceutical composition comprising a mate, and a pharmaceutical composition comprising a vanadium compound selected from vanadil (VO ²⁺ ), metavanadate (VO ₃ ⁻ ) or vanadate (VO ₄ ^3- ) salts, the method of administering these compositions Also included is a pharmaceutical package to be included with this document. Preferably, the composition comprising the vanadium salt is administered prior to the monohydroxyxamate composition. The two components may also be constituted in one composition, for example in the compartment of a capsule, separated by an opaque membrane.

Since the monohydroxyxamate of the formula R-CO-NHOH, in particular Glu (γ) HXM, itself binds to endogenous intracellular vanadium, the vanadium can be modified into an active species that causes a metabolic reaction of insulin. Is a chemical formula R-CO-NHOH, wherein R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n- , (ii) H ₂ N-CH (COOH) -CH ₂ -S-CH ₂ And (iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl, wherein n is 1, 2 or 3 and Y is OH or NH ₂ Also contemplated are pharmaceutical compositions for treating diabetes

In addition, the present invention is used in the manufacture of a pharmaceutical composition for treating diabetes, wherein the formula R-CO-NHOH, wherein R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n- , (ii) H ₂ N-CH (COOH) -CH ₂ -S-CH ₂ -and (iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl, wherein n is 1, 2 or 3, and Y Is OH or NH ₂ ).

In another embodiment, the present invention provides a method for lowering blood glucose concentrations in a diabetic patient, the method comprising: an effective amount of a vanadium complex of a monohydroxyxamate of formula (I) of the present invention, or an effective amount of a chemical formula of the present invention. Vanadium compounds selected from monohydroxyxamate and vanadil (VO ²⁺ ), metavanadate (VO ₃ ⁻ ) or vanadate (VO ₄ ^3- ) salts of R-CO-NHOH, or an effective amount of R- of the present invention A method comprising administering monohydroxyxamate of CO-NHOH alone or in a form combined with insulin treatment.

In another embodiment, the present invention provides a method of inducing normal blood glucose in a diabetic patient, wherein the patient has an effective amount of the vanadium complex of the monohydroxyxamate of formula (I) of the present invention, or an effective amount of the formula R- of the present invention. Vanadium compounds selected from monohydroxyxamate and vanadil (VO ²⁺ ), metavanadate (VO ₃ ⁻ ) or vanadate (VO ₄ ^3- ) salts of CO—NHOH, or an effective amount of R-CO— of the present invention A method comprising administering monohydroxyxamate of NHOH alone or in a form combined with insulin treatment.

Example 1 In Vivo Efficacy of Lipidogenesis Ability of Low Concentrations of VOCl ₂ (10 mM) by Equimolar Monohydroxyxamate.

The following protocol was found to be an in vitro reliable assay, suggesting the potency effect of the amino acid monohydroxyxamate on vanadium (+4) in STZ-rats in vivo.

Activation of lipoogenesis was carried out using 10 μM solutions of various free amino acid monohydroxyxamate (HXM), VOCl ₂ (+4) 10 μM solutions or amino acid-HXM: VOCl ₂ as described in section (c) of the experimental procedure. : 1 complex was performed using 10 μM solution.

The following amino acid monohydroxyxamates were tested: L-glutamic acid γ-monohydroxyxamate [Glu (γ) HXM], glycine hydroxyxamate (Gly-HXM), L-isoleucine hydroxymate (Ile-HXM), L-Tryptophan Hydroxamate (Trp-HXM), L-Tyrosine Hydroxamate (Tyr-HXM), L-cystine Dihydroxamate [Cys (HXM) ₂ ], L-Lysine Hydroxamate (Lys-HXM) , Nicotinic acid hydroxamate (Nic-HXM), L-arginine hydroxamate (Arg-HXM), L-histidine hydroxamate (His-HXM), D-glutamic acid γ-monohydroxamate [D-Glu (γ ) HXM], N-acetyl-L-glutamic acid γ-monohydroxamate [N-acetyl-Glu (γ) HXM], L-aspartic acid β-monohydroxamate [Asp (β) HXM], aminoisobutyric acid Monohydroxyxamate [Aib-HXM].

The results are summarized in Tables I and II. As shown in Table 1, Glu (γ) HXM (10 μM), VOCl ₂ (10 mm) or 1: 1 complexes thereof produced 22%, 40% and 117% of the maximal insulin response, respectively. Thus, the net potentiation effect reached 51%. In addition, Nic-HXM increases the lipidogenic ability of VOCl ₂ (29%, net potentiation effect, see Table 1). Other amino acid hydroxamates tested did not increase the effect of vanadium (+4). The same is true for D-Glu (γ) HXM and N-acetyl-Glu (γ) HXM, indicating that for Glu (γ) HXM, free α-amino groups and L-isomeric forms are essential for increasing efficacy.

Table I. Increased lipid production capacity of low concentrations of VOCl ₂ (10 μM) by equimolar concentrations of amino acid monohydroxyxamate

Maximum Effectiveness Percent of Insulin

Amino acid-HXM alone (10μM) VOCl ₂ alone (10 μM) VOCl ₂ : HXM1: 1 (10 μM) Net potentiation effect (%) Glu (γ) HXM 22% 40% 117% 51% Nic-HXM 11% 40% 80% 29% Aib-HXM 0% (-3%) 40% 22% 0 Lys--HXM 0% (-4%) 40% 36% 0 D-Glu (γ) HXM One% 40% 17% 0 N-acetyl Glu (γ) HXM 0% 40% 24% 0 L-Asp (β) HXM 8% 11 $ 73% 54% Arg-HXM 4% 11 $ 17% About 2% Trp-HXM 2% 11 $ 22% About 9% His-HXM 4% 11 $ 18% About 3%

Table II. Comparison of Insulin Effects of Various VOCl ₂ : HXM (1: 1) and L-Glu (γ) HXM: VOCl ₂ (1: 1)

1: 1 complex of VOCl ₂ with the following HXM Activity on Glu (γ) HXM VOCl ₂ (%) L-Glu (γ) HXM 100% L-Asp (β) HXM, Nic-HXM 70% D-Glue (γ) HXM, N-acetylGlu (γ) HXM, D-Asp (β) HXM, Aib-HXM, Lys-HXM, Arg-HXM, Trp-HXM, His-HXM 0%

Example 2. Comparison of concentration dependent in vitro activation of lipid production by 1: 1 to 1: 5 complexes of Glu (γ) HXM: VOCl ₃ with free VOCl ₂ and free Glu (γ) HXM.

In order to determine the most effective ratio of the Glu (γ) HXM: VOCl ₂ complex that elevates the fetal efficacy of insulin of vanadium, lipid formation is achieved using a 1: 1 to 5: 1 complex, free of Glu (γ) HXM: VOCl ₂ . VOCl ₂ and free Glu (γ) HXM were used as described in section (c) of the experimental procedure. The results shown in FIG. 1 compare the lipid production efficacy of 5mM VOCl ₂ alone and Glu (γ) HXM (5-25 μM) with increasing concentrations and complexed 5mM VOCl ₂ 1: 1 stoichiometry of both materials. The theoretical complex demonstrates that vanadium (+4) was most effective at raising the fetal efficacy of insulin.

Example 3 Effect of Glu (γ) HXM: VOCl ₂ on Blood Glucose Concentration (BGL) of STZ-Rat; Comparison with low concentrations of single VOCl ₂ .

To demonstrate that the vanadium (+4) amino acid monohydroxyxamate chelator increases the normal blood glucose effect of vanadium in vivo, STZ-rats (see section (b) of the experimental procedure) were treated with VOCl ₂ (0.02 mmol / Kg / day) or VOCl ₂ : Glu (γ) HXM 1: 1 complex (0.02 mmol / kg rat / day) was injected intraperitoneally (ip). Blood glucose concentrations were measured over 12 days. The results are shown in FIG. 2, which was shown that intraperitoneal injection of low doses of VOCl ₂ (about 2 mg vanadium / kg / day) alone did not have a significant effect on lowering blood glucose levels, but Glu (γ) HXM: VOCl ₂ 1: 1 complex (about 1 mg vanadium / kg / day) was effective, indicating that the blood glucose concentration of STZ-rat dramatically decreased to normal value. Stable normoglycemia was achieved within 2 days of administration of the complex and lasted for several days after administration (in FIG. 2, dashed lines represent blood glucose concentrations of control normal rats).

The amount of free vanadium (+4) required to induce normal blood glucose is 9.3 mg / kg / day (intraperitoneal administration). Glu (γ) HXM: VOCl ₂ complex (1: 1) reduced the daily dose to about 1 mg / kg / day (intraperitoneal administration). Thus, for this in vivo STZ-rat model, it is believed that the vanadium (+4) potency is increased about 9-fold when vanadium (+4) is complexed to Glu (γ) HXM.

Example 4. Monohydroxyxamate that also raises the fetal efficacy of insulin of vanadium (+5): L-Glu (γ) HXM which highly increases the efficacy of vanadium (+5) to activate glucose metabolism in adipocytes.

To test the synergistic effect of Glu (γ) HXM on complexation with vanadate (+5), lipid formation was performed using a 1: 1 complex of Glu (γ) HXM: VO ₄ ⁺³ (+5), free Na _3. The concentrations of VO ₄ and free Glu (γ) HXM were used at 10 μM to 50 μM, respectively, as described in section (c) of the experimental procedure. Dihydroxa, represented by RL-252 described in US Pat. No. 5,338,759, which increases the fetal ability of vanadium (+4) but is ineffective or even lowers the efficacy of vanadium (+5) in vitro. Unlike the mate chelators, Glu (γ) HXM dramatically increased the insulin-like effect of vanadate (+5) (see FIG. 3). Glu (γ) HXM: VO ₄ ⁺³ (+5) is estimated to be at least 7 times more potent in activating lipid production compared to free Na ₃ VO ₄ or free Glu (γ) HXM.

Example 5. Glu (γ) HXM and Glu (γ) HXM: NaVO ₃ (2: 1) complexes to stimulate hexose influx.

To test the specific effects of Glu (γ) HXM and Glu (γ) HXM: NaVO ₃ (2: 1) complexes on the influx of glucose into cells, ex vivo assays were run on 2-deoxy-D- [6- ³ H] glucose (2-DG) was used. 2-DG is a non-metabolized analog of glucose, whereby this assay shows the effect of the compound on glucose uptake into cells, regardless of glucose metabolism.

Freshly prepared adipocytes (3x10 ⁵ cells / ml) suspended in KRB buffer (pH 7.4) containing 1.0% BSA were treated with insulin (17nM), and Glu (γ) HXM, Glu at the indicated concentrations (20 and 40 μM). Pre-incubation was performed for 30 minutes in the absence and presence of (γ) HXM: NaVO ₃ (2: 1) complex and NaVO ₃ . Aliquots (70 μl) of the above mentioned samples were transferred to a tube containing 2-deoxy-D- [6- ³ H] glucose (0.1 mM final concentration). After 3 minutes, floretine (0.1 nM) was added to terminate 2-DG precipitation in the cells. The sample of suspended cells was then transferred to a tube containing silicone oil, where it was centrifuged to separate the cells from KRB medium and residual 2-DG.

As shown in FIG. 4, the free Glu (γ) HXM and Glu (γ) HXM: NaVO ₃ (2: 1) complexes were found to activate glucose uptake into cells, independent of glucose metabolism. The degree of effect amounted to about 60% and 120% of the maximal insulin response, respectively.

Example 6 Glu (γ) HXM: NaVO ₃ (2: 1) Complexes to Lower Blood Glucose Concentrations of STZ Treated Rats.

To demonstrate that the complex of vanadium (+5) and monohydroxyxamate chelator increases the normal blood glucose effect of vanadium in vivo and to test the normal blood glucose effect of free Glu (γ) HXM in vivo, Diabetic rats were divided into four groups of four to five animals: diabetic control rats; Vanadium (+5) treated rats; Rats treated with Glu (γ) HXM: NaVO ₃ (2: 1) complexes; And rats treated with free Glu (γ) HXM. Each group was injected intraperitoneally with 0.05 mmol / kg of the corresponding compound (at 11 am). As shown in FIG. 5, after the first day (measured blood glucose concentration at 8 am), the blood glucose concentration of the complexed group was lowered to normal concentration.

Example 7 Glu (γ) HXM Activating Lipidogenesis in Rat Adipocytes in the Absence of Externally Added Vanadium

To investigate the normal glycemic efficacy of free Glu (γ) HXM, lipoogenesis was performed as described above using Glu (γ) HXM at a concentration of 10-100 μM. As shown in FIG. 6, Glu (γ) HXM is the only substance of all vanadium binders tested herein that has the ability to produce an insulin effect in the absence of externally added vanadium. L-Glu (γ) HXM is thought to be different from all other amino acids in that small amounts of intracellular vanadium (about 20 nM) can be converted into insulin-like active species.

Example 8 Staurosporin Inhibits Glu (γ) HXM Induced Lipogenesis in Rat Adipocytes. Comparison of Staurosporin Effects on Insulin-Induced Lipogenesis and Vanadate Induced Lipogenesis.

Staurosporin, a potent inhibitor of rat fat CytPTK (ki about 2 nM) and a weak inhibitor of InsRTK (ki about 1 μM), preferentially inhibits the effects of vanadate that stimulates lipid production. To determine if free Glu (γ) HXM also acts via the vanadium pathway, adipocytes were exposed to varying concentrations of staurosporin (as shown in FIG. 7) at 37 ° C. for 30 minutes. Lipogenesis was then performed using adipocytes prepared as described above in the presence of insulin (17 nM), sodium metavanadate (0.8 mM) or Glu (γ) HXM (100 μM). Maximal activation (100%) is obtained with insulin, vanadate or Glu (γ) HXM in the absence of staurosporin.

FIG. 7 shows the degree of lipoogenesis induced by free Glu (γ) HXM, sodium metavanadate or insulin with increasing concentrations of staurosporin. Activation of lipoogenesis by Glu (γ) HXM was inhibited in a dose dependent manner by staurosporin. Inhibition curves were similar to those obtained for vanadate induced lipidogenesis (rather than those obtained for insulin induced lipidogenesis), suggesting that free Glu (γ) HXM acts through the vanadium (insulin-dependent) pathway. do.

Example 9 Lipogenesis: Comparison of Normal Adipocytes with Vanadium Rich Adipocytes Treated with Glu (γ) HXM.

Free Glu (γ) HXM is unique in that it activates lipoogenesis in rat adipocytes in the absence of externally added vanadium. This effect is evident through the vanadium pathway by experimental data suggesting that free Glu (γ) HXM can convert physiologically small amounts of vanadium endogenously present in rat adipocytes into the native active species of insulin. Proven herein.

To demonstrate this effect, male Wistar rats were injected subcutaneously (sc) with NaVO ₃ (12 mg / kg / day) daily for 5 days (hereinafter referred to as “vanadium rich rats”). Lipidogenesis was performed as described above using free Glu (γ) HXM to compare freshly prepared rat adipocytes (3 × 10 ⁵ cells / ml) from normal and vanadium rich rats. As shown in FIG. 8, free Glu (γ) HXM has been shown to increase the effect of intracellular vanadium to a much greater extent in vanadium rich rat cells.

Example 10 Vanadium Complexes of Stable Glu (γ) HXM.

Glu (γ) HXM this, in order to demonstrate that the formation of stable complexes with NaVO ₃ and VOCl ₂ to maintain a highly active for an extended period of time when allowed to stand as a dry powder at room temperature, Glu (γ) HXM: NaVO 3 and 2: 1 complexes of Glu (γ) HXM: VOCl ₂ were prepared by dissolving monohydroxyxamate and vanadium salts in water at 2: 1 equimolar concentrations, respectively. The aqueous solution was then mixed, frozen with liquid nitrogen and lyophilized. The dried powder obtained was left at room temperature for 4 weeks. Lipidogenesis was then performed using the free Glu (γ) HXM, NaVO ₃ and VOCl ₂ , and Glu (γ) HXM: NaVO ₃ and Glu (γ) HXM: VOCl ₂ 2: 1 complexes as dry powders. It was performed as described.

references

Brichard, S. M., and Henquin, J. C. (1995) Trends in Pharmacol. Sci. 16, 265-270.

Brown, D.A., R.A. Coogan, N.J.Fitzpatrick, W.K. Glass, D.E. Abukshima, L. Shiels, M. Ahlgren, K. Smolander, T.t. Pakkanen, T.A. Pakkanen and M. Perakyla (1996) J. Chem. Soc., Perkin Trans. 2, 2673-2679.

Cohen, N., Halberstam, M., Shimovich, P., Chang, C.R., Shamoon, H., and Rossetti, L. (1995) J. Clin. Invest. 95, 2051-2509.

Li, J., Elberg, G., Crans, D. C., and Shechter, Y. (1996) Biochemistry 35, 8314-8318.

Moody, A. Sta, M, A., Stan, M., and Gliemann, J. (1974) Horm. Metab. Res. 6, 12-16.

Rodbell, M (1964) J. Biol. Chem. 239, 375-80.

Sakurai et al. (1995) BBRC, 214, 1095-1101.

Shechter, Y., Shisheva, A., Lazar, R., Libman, J. and Shanzer, A. (1992) Biochemistry 31, 2063-68.

Shechter, Y., Li, J., Meyerovitch, J., Gefel, D., Bruck, R., Elberg, G., Miller, D. S., and Shisheva, A., (1995) Molec. Cell. Biochem. 153, 39-47.

Tournaire, R., S. Malley, F. Hamedi-Sangsari, N. Thomasset, J. Grange, J.F. Dore and J. Vila, (1994) Int J. Cancer 58, 420-425.

Vila, J., N. Thomasset, C. Navarro and J.F. Dore (1990) Int. J. Cancer 45, 737-743.

Claims (20)

Monohydroxyxamate vanadium complexes of formula (I) R-CO-NHOH.X (I) Where R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n -where n is 1, 2 or 3 and Y is OH or NH ₂ , (ii) H ₂ N—CH (COOH) —CH ₂ —S—CH ₂ — and (iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl; X is a vanadium compound selected from vanadil (VO ²⁺ ), metavanadate (VO ₃ ⁻ ) or vanadate (VO ₄ ^3- ) salts. The monohydroxyxamate vanadium complex of claim 1 which is a 1: 1 or 2: 1 vanadil complex of L-glutamic acid γ-monohydroxamate of the formula: H ₂ N-CH (COOH) -CH ₂ -CH ₂ -CO-NHOH: VOCl ₂ A monohydroxyxamate vanadium complex according to claim 1 which is a 1: 1 or 2: 1 vanadate complex of L-glutamic acid γ-monohydroxyxamate of the formula: H ₂ N-CH (COOH) -CH ₂ -CH ₂ -CO-NHOH: NaVO ₃ The monohydroxyxamate vanadium complex of claim 1 which is a vanadil complex of L-aspartic acid β-monohydroxamate of the formula: H ₂ N-CH (COOH) -CH ₂ -CO-NHOH: VOCl ₂ The monohydroxyxamate vanadium complex of claim 1 which is a vanadil complex of nicotinic acid hydroxyxamate of the formula: Pyridyl-3-CO-NHOH: VOCl ₂ A pharmaceutical composition comprising an effective amount of the vanadium complex of the monohydroxyxamate according to any one of claims 1 to 5 as an active ingredient, optionally together with a pharmaceutically acceptable carrier. 7. A pharmaceutical composition according to claim 6, which is useful for diabetic patients to lower blood glucose levels and / or induce normal blood glucose. 8. A pharmaceutical composition according to claim 7, which is in a form suitable for oral administration. A vanadium compound selected from vanadil, metavanadate and vanadate salts, and of the formula R-CO-NHOH, wherein R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n- , (ii) H ₂ N-CH (COOH) -CH ₂ -S-CH ₂ -and (iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl, wherein n is 1, 2 or 3, and Y Is OH or NH ₂ ) a pharmaceutical composition for treating diabetes comprising monohydroxyxamate, A pharmaceutical composition wherein the vanadium salt and the monohydroxyxamate are separated from each other in the composition. A first compartment containing a vanadium compound selected from vanadil, metavanadate and vanadate salts, and a formula R-CO-NHOH, wherein R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n -, (ii) H ₂ N-CH (COOH) -CH ₂ -S-CH ₂ -and (iii) pyridyl, piperidyl or tetrahydroisoquinolinyl, wherein n is 1, 2 Or 3 and Y is OH or NH ₂ ) two compartments of a second compartment containing a monohydroxyxamate; And instructions for administering these two compartments to lower blood glucose levels and / or induce normal blood glucose in diabetic patients. Use of the vanadium complex of the monohydroxyxamate according to any one of claims 1 to 5 for the preparation of a pharmaceutical composition useful for lowering blood glucose levels and / or inducing normal blood glucose in diabetic patients. Formula R-CO-NHOH, wherein R is (i) H ₂ N-CH (COY)-(used to prepare pharmaceutical compositions useful for lowering blood glucose levels and / or inducing normal blood glucose in diabetic patients) CH ₂ ) _n- , (ii) H ₂ N-CH (COOH) -CH ₂ -S-CH ₂ -and (iii) pyridyl, piperidyl or tetrahydroisoquinolinyl, wherein n Is 1, 2 or 3, and Y is OH or NH ₂ ). A method of lowering blood glucose levels and / or inducing normal blood glucose in a diabetic patient, the method comprising administering to the patient an effective amount of a vanadium complex of monohydroxyxamate of formula (I): R-CO-NHOH.X (I) Where R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n -where n is 1, 2 or 3 and Y is OH or NH ₂ , (ii) H ₂ N—CH (COOH) —CH ₂ —S—CH ₂ — and (iii) a residue selected from pyridyl, piperidyl or tetrahydroisoquinolinyl; X is a vanadium compound selected from vanadil (VO ²⁺ ), metavanadate (VO ₃ ⁻ ) or vanadate (VO ₄ ^3- ) salts. The method of claim 13, wherein the vanadium complex is a 1: 1 or 2: 1 vanadil complex of L-glutamic acid γ-monohydroxamate of the formula: H ₂ N-CH (COOH) -CH ₂ -CH ₂ -CO-NHOH: VOCl ₂ The method of claim 13, wherein the vanadium complex is a 1: 1 or 2: 1 vanadate complex of L-glutamic acid γ-monohydroxamate of the formula: H ₂ N-CH (COOH) -CH ₂ -CH ₂ -CO-NHOH: NaVO ₃ The monohydroxyxamate vanadium complex of claim 13 wherein the vanadium complex is a vanadil complex of β-aspartic acid monohydroxyxamate of the formula: H ₂ N-CH (COOH) -CH ₂ -CO-NHOH: VOCl ₂ A method of lowering blood glucose levels and / or inducing normal blood glucose in a diabetic patient, the method comprising the vanadium compound selected from an effective amount of vanadil, metavanadate and vanadate salts, and an effective amount of the formula R-CO-NHOH, wherein , R is (i) H ₂ N-CH (COY)-(CH ₂ ) _n- , (ii) H ₂ N-CH (COOH) -CH ₂ -S-CH ₂ -and (iii) pyridyl, p Selected from ferridyl or tetrahydroisoquinolinyl, wherein n is 1, 2 or 3 and Y is OH or NH ₂ . 18. The method of claim 17, wherein the vanadium compound is administered prior to the monohydroxyxamate compound. The method of claim 13, wherein the vanadium complex is administered orally. The method of claim 13, wherein the treatment is combined with administration of insulin.